A new strategy: identification of specific antibodies for neutralizing epitope on SARS-CoV-2 S protein by LC-MS/MS combined with immune repertoire

Although the SARS-CoV-2 vaccine has been widely used worldwide, not all individuals can produce neutralization antibodies, so it is still urgent to find and prepare neutralization antibodies for COVID-19 prevention or treatment. In this study, we created a new strategy to effectively obtain neutralizing antibodies or complementary determining region 3 (CDR3) of neutralizing antibodies against SARS-CoV-2. We first predicted and synthesized several B cell epitopes on RBD and adjacent RBD of S protein, then the B cell epitopes were used to prepare affinity chromatography columns respectively and purify the binding IgG from serum samples of convalescent COVID-19 patients. After these IgGs were identified to have neutralizing activity, the peptide sequences of the antigen-binding regions (variable region) of neutralizing antibodies were analyzed by protein mass spectrometry. Subsequently, the B cells from the same individual were sorted and used to obtain their full BCR repertoire by 5′ RACE combined with high-throughput of PacBio sequencing method. Then, the peptide sequence of neutralizing antibody variable region by protein mass spectrometry was mapped to the full BCR repertoire and found the full variable region sequence of neutralizing antibodies. Finally, we obtained and synthesized numerous CDR3 peptides of neutralizing antibodies to confirm the neutralizing activity for SARS-CoV-2 infection. Our results indicate that the novel scheme will be suitable for rapid screening of neutralizing antibodies, including screening neutralizing antibodies against SARS-CoV-2 and other pathogenic microorganisms. Supplementary Information The online version contains supplementary material available at 10.1186/s43556-022-00085-0.


B Cell Epitope Prediction
SARS-CoV-2 genome sequence and protein annotation were from Wuhan-Hu-1 isolate (GenBank accession number: MN908947). Linear B cell epitopes based on the spike protein antigen sequence characteristics using amino acid scales and hidden Markov models. Briefly, the methods of BepiPred 1.0 and BepiPred 2.0 were combined with default thresholds via the portal website (http://tools.iedb.org/bcell/). BepiPred 1.0 predicts the location of linear B-cell epitopes using a combination of a hidden Markov model and a propensity scale method (http://www.cbs.dtu.dk/services/BepiPred-1.0/). In contrast, BepiPred 2.0 predicts B-cell epitopes using a Random Forest algorithm trained on epitopes and non-epitope amino acids determined from crystal structures 17 . Because neutralizing antibodies were strongly expected, the epitopes in the receptor-binding motif (amino acids 438-506 in the full protein sequence) 18 and the adjacent region were paid more attention. The predicted epitopes were also checked the surface accessibility using the website 'Emini Surface Accessibility Prediction' method. We selected four linear B cell epitopes, including LFRKSNLKPFERDISTE (aa. 455-47, E1), E2), E3), and QQFGRDIADTTDAVRDPQ (aa. 563-580, E4) for preferred peptide synthesis, the E1-3 are located on the receptor-binding domain (RBD), E4 is located on adjacent RBD. Then, the epitope peptides were synthesized by the Chinese Peptide Company，respectively. It is worth noting that since both E2 and E3 are short moreover, the distance between the two epitopes is very close. E2 and E3 are synthesized in series.

Purification of epitope peptide-specific IgG
E1, E2-E3,E4 peptide and S protein (Purchase from OkayBio) were coupled to CNBractivated Sepharose 4FF (17098101, GE Healthcare, Chicago, IL, USA) according to the manufacturer's recommendation. Briefly, the preactivated Sepharose 4FF was suspended in 1mM HCl for 30 min on ice and washed with cold coupling buffer (0.1 mol/L NaHCO3, 0.5 mol/L NaCl, pH 8.3). Then dissolve the peptide in coupling buffer and add to the washed sepharose and incubate overnight at 4 °C. Wash and resuspend the coupled gel in 0.1mol/L Tris-HCl (pH 8.0) for 2 hours at room temperature to block unused activated sites. Then wash the gel six times with alternating 0.1 mol/L Tris, 0.5 mol/L NaCl, pH 8.0 and 0.1 mol/L NaAc, 0.5 mol/L NaCl, pH 4 buffers and then PBS. Next, the purified IgG from each COVID-19 patient was first incubated with E1-coupled Sepharose column overnight at 4 °C, then the suspension was transferred to E2/E3-coupled Sepharose column and incubation for 2 hours at room temperature (RT), the solution was collected and transferred to E4-coupled Sepharose column and incubation for 2 hours, RT. Finally, the suspension transferred to the S1-coupled Sepharose column. All columns as above were washed with PBS and eluted using 0.1 mol/L Glycine, pH 2.4 to obtain specific IgG for the E1, E2/3, E4, and other epitopes on S1 protein, respectively, and soon neutralized by 1 mol/L Tris, pH 8.0, and exchange to PBS by Amicon Ultra centrifugal filter.

Pseudovirus neutralization assay
The pseudovirus neutralization assays were performed using hACE2-expressed HEK293 cell lines (purchase from PackGene Biotech). 5×10 4 /well cells were added to the well and cultured at 37 ℃ for 8 hours. The synthesized CDR3 peptides, IgG specifically bind to either E1-E4 or S1 protein, and sera were dissolved in DMEM complete medium and mixed with SARS-CoV-2 pseudovirus (purchased from Fubio Biological Technology Company, Pseudovirus-2019-nCOV) with a TCID50 of 5×10 4 TU in a 1.5 ml Eppendorf tube with a 100μL final volume (peptides at a final concentration 10 μg/μL, IgG at a final concentration 1 μg/μL, sera was diluted 160x) and incubated for one hour at 37 ℃. Negative control tube was supplied with 100 μL DMEM. Positive control tube was supplied with 100 μL DMEM containing SARS-CoV-2 pseudovirus. After the cells adhered to the wall, the supernatant was taken out from each well, and a 100 μL mixed solution of virus and peptides/antibodies was added to the well, and the two control groups were treated with the same operation. 48-well plate was cultured for 18 hours at 37 ℃ supplied with 5% CO2. The cells were directly washed with PBS and centrifuged at 3000rpm, and the supernatant was discarded. 200 μL PBS was added, and the GFP-FITC was detected by flow cytometry. The neutralization efficiency of peptides/antibodies against SARS-CoV-2 pseudovirus was determined as [(Positive control MFI-experimental MFI)/(Positive control MFI-Negative control MFI)] × 100.

SDS-PAGE and LC-Mass/Mass
Purified IgG with neutralizing antibodies potential was denatured with heat, analyzed by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and stained with Coomassie Brilliant Blue. Cut at 55kD and 25kD, which are IgG heavy chain and light chain positions respectively, and send to the Proteome Analysis Platform of the Peking University Medical Department and Health Analysis Center.

Enrichment of B cells from PBMC
B cells were isolated from fresh or previously frozen PBMCs by immunomagnetic positive selection according to the manufacturer's protocol (EasySep™ Human CD19 Positive Selection Kit II, STEMCELL, Vancouver, Canada). Purified B cells were isolated and washed with PBS containing 2% (v/v) fetal bovine serum (FBS) and 1 mM EDTA.

RNA extraction and cDNA synthesized by 5'-RACE
According to the manufacturer's instructions for sorted B cells, total RNA was extracted using the RaPure Total RNA Micro Kit (Magen, Guangzhou, China), then the cDNA was synthesized by 5'-RACE using SMARTer® RACE 5'/3' Kit (Takara Bio Inc., Shiga, Japan) and generated a complete cDNA copy with the additional specific sequence at the 5' end.

Amplification of BCR transcripts with barcoded primers
Nested PCR was used to amplify the variable regions of Ig. The upstream primer targeted an additional specific sequence from 5'-RACE, and the downstream primer targeted constantregion for IGHG, IGHA, IGHM, IGHD, IGK, and IGL in both of first-round and second-round PCR. Especially, barcodes were added to the second-round PCR primers that is convenient to distinguish BCRs from a different individual. PCR program for both rounds were: 5 cycles at 94 °C for the 30s, 5 cycles at 68 °C for 30s, 25 cycles at 72 °C for 3 minutes (first-round PCR) and 40 cycles at 94 °C for 30s, 68 °C for 30s, and 72 °C for 2 minutes (second-round PCR).
The amplified DNA products were recovered from the agarose gel using a DNA Recovery Kit and sent to Novogene company for sequencing.

Sequencing and barcode filtering
Sequencing libraries were prepared using PacBio sequencing. Raw reads were retained only if there are the sequences contained in the barcode. Constant region with highest sequence similarity was identified by matching to the reference constant region sequences from the IMGT database 19 and sequences were trimmed to give only the variable (VDJ) regions.
Sequences with significant similarity to reference IGHV, D and J genes from the IMGT database using BLAST were retained 20 . Ig gene usages and sequence annotation were performed in IMGT V-QUEST, where repertoire differences were performed by custom scripts in Python.

BCR repertoire analysis
Applying for an account in the IMGT database, login and use IMGT/HighV-QUEST (version 1.7.1) to submit the sequencing data to the IMGT database. Download the completed data, which were zip files, and decompress it into the folders. To get the CDR3 sequence and full length of variable region sequence corresponding to V(D)J usage in all samples by running scripts.

BCR sequencing and Mass spectrometric sequence alignment
The mass spectrometry peptide sequences of IgG that respectively bound to E1, E2-E3, E4, and S1 protein, were used to map to the variable region amino acid sequences obtained by BCR repertoire analysis from the same individual to obtain the complete sequence of IgG heavy chain and light chain variable region with neutralization potential. Moreover, we also try to find whether there were the same or similar IgG sequences with neutralization potential among different individuals. The analysis was completed with MaxQuant software.

The binding ability of CDR3 peptides of IgG with neutralizing potential to S1 protein analyzed by microscale thermophoresis
Since antibodies recognize antigens mainly depend on the CDR3 region, we synthesize 57 CDR3 peptides of these heavy chains and light chains with neutralizing potential and verify their binding ability to S1 protein analyzed by microscale thermophoresis (MST). Briefly, single-cycle kinetics experiments with a Biacore T200 instrument (GE Healthcare) was used to analyze the binding of the S1 protein to the various CDR3 sequence. Purified S1 protein was first immobilized on a series S sensor chip protein A (GE Healthcare) at 800-1200 response units (RU) in PBS containing 0.02% sodium azide. One cell on the sensor chip was empty to serve as a blank. Then, a series of concentrations (i.e., 0.8, 4, 20, 100, and 500 nM) of soluble CDR3 peptide was injected in PBS at a flow rate of 60 μL/min. The sensor chip was regenerated using 10 mM Glycine-HCl (pH=1.5) buffer. A 1:1 binding model was used to describe the experimental data. Due to conformational change in these cases, we fitted a two-state binding model that assumes two binding constants.

Focus reduction neutralization test (FRNT)
FRNT assay was used for the evaluation of the Abs (peptide) neutralization effect. Vero E6 cells were seeded into a 96-well plate one day before infection. The next day, two-serially diluted Abs (peptide) and SARS-CoV-2 (80-120 FFU) were combined in DMEM (2% FBS) and incubated at 37 ℃ for 1 hour, then 50μl mixtures were added into 96-well plate seeded with Vero E6 cells and incubated in 37 ℃ for 1 hour with rocking every 15 min. Then mixtures were removed and 100 μl MEM containing 1.2% Carboxymethylcellulose (1.2% CMC) was added. The medium was discarded after 24-hour post-infection, and the cell monolayer was fixed with 4% paraformaldehyde buffer at RT for 2 hours and permeabilized with 0.2% Triton X-100 for 20 min. Then the plates were sequentially stained with rabbit anti-SARS-CoV-2 N IgG (Cat. No. 40143-T62, Sino Biological Inc) and HRP-conjugated goat anti-rabbit IgG(H+L) (No.109-035-088, Jackson ImmunoResearch) at 37 ℃ for 1 hour respectively. The reactions were developed with KPL TrueBlue Peroxidase substrates and CTL ImmunoSpot S6 Ultra reader (Cellular Technology Ltd) was used to calculate the numbers of SARS-CoV-2 foci. The half-maximal inhibitory concentration (IC50) was determined by 50% focus reduction neutralization test titers (FRNT50) used to evaluate the potency of Abs in inhibiting SARS-CoV-2 replication.

IFA neutralization assay
To determine whether Abs (peptide) could neutralize the infection of SARS-CoV-2, IFA neutralization assay was performed. Vero E6 cells were seeded into a 96-well plate one day before infection. Then, serial 2-fold diluted peptides were mixed with quantitative SARS-CoV-2 (MOI=0.01) in microplates at 37 °C for 1 hour. Then the sample-virus mixture was transferred to the confluent cell monolayer in duplicate and incubated at a multiplicity of infection (MOI) of 0.01 at 37 °C for 24h. After fixation with 4% paraformaldehyde, the monolayers were permeabilized with 0.2% triton X-100, followed by a 1 h incubation at 37 °C with the crossreactive rabbit anti-SARS-CoV-2 N IgG (Sino Biological Inc) as the primary antibody. Then the cells were washed with PBS and incubated with Alexa Fluor 488 conjugated goat anti-rabbit IgG (Invitrogen) as the secondary antibody. Cells were washed twice with PBS and nuclei were stained with DAPI (Invitrogen, Germany) for 15 min at room temperature.

Statistical Analysis
All data were analyzed by normality and lognormality tests to identify whether the data belong to a normal distribution, which was decided by the p-value of the Shapiro-Wilk test.

Supplementary tables and Figures
Supplementary Figure 1. Ability of blocking pseudovirus in purified antibodies a. E1, E2-E3, E4, and S1 protein-specific IgGs purified by affinity column from 3 healthy subjects (HS) and 15 COVID-19 convalescents have been detected the ability to block pseudovirus by flow cytometry as described in Fig 2c. B. E1, E2-E3, E4, and S1 protein-specific IgGs purified by affinity column from healthy subjects (HS) and COVID-19 convalescents have been detected the ability to block pseudovirus by luciferase assay.